Manuscript Click here to download Manuscript: 15-00389 Bengoa and Kypta revised.docx Click here to view linked References 1 Canonical and Noncanonical Wnt Signaling in Neural 2 Stem/Progenitor Cells 3 4 1* 1, 2 5 Nora Bengoa-Vergniory and Robert M. Kypta 6 7 8 9 1 Cell Biology and Stem Cells Unit, CIC bioGUNE, Bilbao, Spain 10 11 12 2 Department of Surgery and Cancer, Imperial College London, London, UK 13 14 15 * Present address Department of Physiology, Anatomy and Genetics, Oxford 16 17 University, UK 18 19 20 *Correspondence: [email protected], [email protected], 21 22 [email protected] 23 24 25 26 Running title: Wnts in Neural Stem Cells 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Abstract 1 2 3 4 5 The first mammalian Wnt to be discovered, Wnt-1, was found to be essential for 6 7 the development of a large part of the mouse brain over 25 years ago. We have 8 9 10 since learned that Wnt family secreted glycolipoproteins, of which there are 11 12 nineteen, activate a diverse network of signals that are particularly important 13 14 15 during embryonic development and tissue regeneration. Wnt signals in the 16 17 developing and adult brain can drive neural stem cell self-renewal, expansion, 18 19 20 asymmetric cell division, maturation and differentiation. The molecular events that 21 22 take place after a Wnt binds to its cell-surface receptors are complex and, at times, 23 24 25 controversial. A deeper understanding of these events is anticipated to lead to 26 27 improvements in the treatment of neurodegenerative diseases and stem cell-based 28 29 30 replacement therapies. Here we review the roles played by Wnts in neural stem 31 32 cells in the developing mouse brain, at neurogenic sites of the adult mouse and in 33 34 35 neural stem cell culture models. 36 37 38 39 40 Keywords: Wnt signaling, neural stem cells, beta-catenin, AP-1 family transcription 41 factors 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 2 65 1 Wnt signaling 2 3 4 Wnt proteins play roles in many cellular and physiological processes, regulating 5 6 7 cell proliferation, differentiation, migration and patterning during embryonic 8 9 development and tissue homeostasis in the adult [1, 2]. The Wnt family in 10 11 12 mammals is comprised of nineteen secreted glycolipoproteins that are able to bind 13 14 to a wide variety of receptors and elicit a number of different responses in the cell 15 16 17 [3]. These have classically been divided into canonical (b-catenin-dependent) and 18 19 noncanonical (b-catenin-independent) Wnt signaling pathways. 20 21 22 23 Canonical Wnt signaling 24 25 In the canonical Wnt signaling pathway, b-catenin is actively degraded by a protein 26 27 28 complex that includes Axin, glycogen synthase kinase-3 (GSK-3), casein kinase 1 29 30 (CK1) and adenomatous polyposis coli (APC). In the classical pathway, binding of a 31 32 33 Wnt protein to receptors of the frizzled (FZD) and low-density lipoprotein 34 35 receptor-related protein (LRP5/6) families leads to membrane recruitment of Axin 36 37 38 and disheveled (DVL), thereby disrupting the function of the degradation complex. 39 40 b-catenin is thus stabilized, enters the nucleus and activates genes in association 41 42 43 with T-cell factor/lymphoid enhancer factor-1 (TCF/LEF) family transcription 44 45 factors [1]. However, a more recent version of the model (Fig. 1) posits that the 46 47 48 destruction complex is not disrupted by Wnt activation and that changes in the 49 50 levels of free and transcriptionally active b-catenin result from relocation of the 51 52 53 complex to the membrane, which disrupts b-catenin ubiquitination rather that the 54 55 56 complex itself, leading to b-catenin accumulation [4]. Other extracellular ligands 57 58 have been shown to alter the output of the pathway. Members of the Dickkopf and 59 60 61 62 63 64 3 65 sFRP families, and WIF1 and Cerberus can bind to Wnt receptors or Wnt ligands 1 2 inhibiting or, in some instances, enhancing their effects [5, 6]. In addition, R- 3 4 5 spondins (RSPOs) modulate the Wnt response at the cell membrane by binding to 6 7 leucine-rich repeat containing G protein-coupled receptors (LGRs) [3]. 8 9 10 The canonical pathway is implicated in many human diseases [2]. Perturbations 11 12 in the levels of Axin, APC, β-catenin, LEF1 or TCF4, for example, contribute to the 13 14 15 initiation and/or progression of several different types of cancer [7–12]. It is 16 17 therefore not surprising that so much effort has gone into the development of new 18 19 20 drugs based on our knowledge of Wnt signaling to treat disease. Among the 21 22 commercially available drugs that have been developed to manipulate Wnt 23 24 25 signaling are IWP‐2 and Wnt-C59, potent porcupine inhibitors that block Wnt 26 27 secretion [13, 14], IWR‐1 and XAV939, which are tankyrase inhibitors that 28 29 30 stabilize Axin and thereby inhibiting canonical Wnt signaling [13, 15], CHIR99021 31 32 (CHIR), a GSK-3 inhibitor that activates Wnt signaling [16], and iCRT-14, which 33 34 35 inhibits the β-catenin/TCF complex [17]. These inhibitors, as well as several others 36 37 not mentioned here, have been important for driving progress of research in this 38 39 40 field, and the development of possible therapies for Wnt-related diseases. 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 4 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Figure 1. Representation of canonical Wnt signaling, according to Li et al. [4]. 32 33 34 Wnt binds to FZD receptors and LRP5/6 co-receptors, recruiting the destruction 35 36 complex to the membrane, thereby preventing β-catenin (β-CAT) ubiquitination, 37 38 39 leading to its accumulation and entry into the nucleus; ubiquitin, UB; nucleus, NUC. 40 41 42 Noncanonical Wnt signaling 43 44 45 Noncanonical Wnt signals (Fig. 2) regulate DVL and other intracellular proteins to 46 47 activate the Planar Cell Polarity (PCP) pathway, the Wnt-Calcium (Ca2+) pathway 48 49 50 and other b-catenin/TCF-independent events [18]. In the Wnt-PCP pathway, FZD 51 52 receptors activate a signaling cascade that involves the small GTPases Rho and Rac 53 54 55 and c-Jun N-terminal kinase (JNK), leading to changes in the cytoskeleton and 56 57 58 activation of Activator Protein-1 (AP-1) family transcription factors [19]. Non- 59 60 canonical Wnt stimuli induce association of DVL associated activator of 61 62 63 64 5 65 morphogenesis (DAAM) proteins with FZD, DVL and GTP-bound Rho, which is then 1 2 able to activate Rho-associated, coiled-coil containing protein kinase (ROCK) and 3 4 5 even JNK. Although JNK is generally associated with phosphorylation and 6 7 activation of c-Jun, it phosphorylates many other proteins, including activating 8 9 10 transcription factor 2 (ATF2) and cyclic AMP response element-binding protein 11 12 (CREB), which heterodimerize with c-Jun and other AP-1 family members to alter 13 14 15 gene expression [20]. This pathway regulates cell polarity in several 16 17 morphogenetic processes in vertebrates, including gastrulation, neural tube 18 19 20 closure and orientation of stereocilia in the inner ear [19]. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Figure 2. Schematic representation of noncanonical Wnt signaling 56 57 Noncanonical Wnt signals activate several different pathways through distinct 58 59 intracellular effectors. In the PCP pathway, Wnt activates Rho kinase (ROCK) and 60 61 62 63 64 6 65 JNK, thereby eliciting changes in gene expression and remodeling of the 1 2 cytoskeleton. In the Wnt-Calcium pathway, DVL activation triggers activation of 3 2+ 4 protein kinase C (PKC) and Ca release. This then activates nuclear factor of 5 activated T-cells (NFAT)-dependent gene expression, with concomitant inhibition 6 7 of b-CAT. Receptor tyrosine kinases (RTKs) activate phosphoinositide-3 kinase 8 9 (PI3K), which can result in JNK activation. FZD receptors can activate several 10 11 intracellular effectors, including protein kinase A (PKA) and p38 kinases. 12 13 Syndecan, SYN; dishevelled associated activator of morphogenesis 1, DAAM1; 14 15 Calcineurin, CNA; calmodulin kinase II, CAMKII; phospholipase C, PLC; adenylate 16 17 cyclase, AC; mitogen-activated protein kinase kinase, MKK. 18 19 20 21 In the Wnt-Ca2+ pathway, Wnt binding to FZD receptors activates DVL, leading to 22 23 24 activation of phospholipase C (PLC), producing 1,2 diacylglycerol (DAG), which 25 26 activates protein kinase C (PKC), and inositol 1,4,5-tri-phosphate (IP3), which 27 28 activates calcium release from the endoplasmic reticulum. Other events such as 29 30 31 activation of ROCK have also been linked to this pathway [21].